This is a proposal to study very early steps in the formation of U1 small nuclear RNA (U1 RNA). Precursors of U1 (pre- U1RNA) and other snRNAs that are made by RNA polymerase II are exported from the nucleus to the cytoplasm where they undergo incorporation into small nuclear ribonucleo- protein particles (snRNPs), base modification and trimming at the 3' end. These snRNPs are then imported into the nucleus, where final modification of the RNA occurs and where they participate in splicing of messenger RNA precursors. Without appropriate formation of snRNPs, cells would die since they would be unable to process messenger RNA precursors. The three goals of this project are: 1) To define mechanisms by which cells control the types and amounts of U1 RNAs. We will do this in mouse tissue culture cells that have been transfected either stably or transiently with mouse chimeric U1RNA genes. These experiments are designed to test whether the cells control U1RNA levels by limiting synthesis or degradation of RNA and to learn if these amounts are monitored by sensing the levels accumulated products. 2) To define complexes that contain pre-U1 RNA prior to export of the RNA to the cytoplasm. We plan to do this in Xenopus laevis oocytes or oocyte nuclei (germinal vesicles). We will analyze the proteins with which pre- U1RNA interacts prior to export by purification of the 10-11S particles containing pre-U1RNA, photo cross lining of RNA and proteins, and by analysis of particles containing mutated RNA. 3) To learn how pre-U1RNA is exported. For this, we will exploit our ability to isolate intact, functioning nuclei (germinal vesicles) to develop a defined experimental system for the analysis of export. This will allow us to test if cytoplasm is required and if nuclear proteins accompany the RNA through the nuclear pore complex. The participation of various nuclear and cytoplasmic structures will be studied by cell fractionation, electron microscopy and isolation of RNA- protein complexes.